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    <title>Implementing LRE-based Assays</title>
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        <h1 align="center">Implementing LRE-based Assays</h1>
		<p align="left"><b>LRE was developed for SYBR Green I-based assays<br>
		</b>It is important to note that although LRE qPCR has been found to be 
		effective for a number of enzyme formulations that use SYBR Green I 
		detection, 
		other fluorescence chemistries, particularly probed-based assays, may 
		not be effective. </p>
		<p align="left"><b>Do not use clear tubes or plates<br>
		</b>As discussed in the <a href="../quality_control/optical_precision_overview.html">optical 
		precision</a> overview, maximizing the optical precision is essential 
		for effective implementation of LRE. White plates and tubes have been 
		found to increase the fluorescence intensity by about 5X above that for 
		generated by clear tubes or plates. This large increase in fluorescence 
		intensity greatly increases the performance of an instrument&#39;s optical 
		system, which in most cases, overcomes the limitations of implementing 
		LRE for instruments that generate low optical precision. </p>
		<p align="left"><b>Setting the minimum Fc can increase quantitative 
		performance<br>
		</b>To maintain general compatibility with the large range of optic 
		scales generated by different instruments, the minimum Fc (minFc) 
		defaults to one cycle below C<sub>1/2</sub>. However, quantitative 
		accuracy can be increased by empirically selecting a minFc within the 
		lower region of the profiles, as is described in the
		<a href="../lre_window_selection/automated_window_selection.html">
		Automated Window Selection</a> section.</p>
		<p align="left"><b>Some enzyme formulations produce anomalous 
		amplification kinetics<br>
		</b>As described in the
		<a href="../issues/profile_arcing.html">profile arcing section</a>
		section, some enzyme formulations, such as ABI&#39;s AmpliTaq Gold, have been found to 
		produce extensive &quot;profile arcing&quot;, which does not conform 
		well to the 
		LRE model. Enzyme formations that have been found to be effective for 
		LRE analysis include Qiagen QuantiTect, Agilent Brilliant II, and 
		Invitrogen Platinum SYBR, although many other SYBR Green I-based 
		formulations are also likely to be 
		effective.</p>
		<p align="left"><b>Conducting performance benchmarks<br>
		</b>One of the most effective methods for testing the 
		performance of an assay and/or instrument is to run a standard optical 
		calibration reaction set, which is normally used for optical 
		calibration. As described in the
		<a href="../quality_control/developing_performance_benchmarks.html">
		developing performance benchmarks</a> section, the 
		universal nature of lambda gDNA can provide a foundation from which to 
		assess the general performance of any qPCR assay.</p>
		<p><b>Assessing quantitative accuracy<br>
		</b>As is true for any quantitative technology, assessing quantitative 
		accuracy should be an important objective during implementation of a new 
		qPCR assay. As discussed in the
		<a href="../quality_control/quality_control.html">quality 
		control section</a>, Poisson distribution allows absolute quantification 
		to be conducted, independent of the kinetic 
		and optical parameters upon which real-time PCR is based. </p>
		<p>Referred to 
		as &quot;<a href="../quantitative_accuracy/lda_overview.html">limited 
		dilution assay</a>&quot;, the analysis simply requires that the target be 
		diluted to about 0.7 molecules per aliquot and 16-32 replicate 
		amplification reactions be conduced. Target quantity is then determined from the number of 
		nil reactions that result from aliquots lacking any 
		target molecules.</p>
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